Error with assembleCells preventing the 10x-sc-xcr-vdj pipeline from compeleting

[Khang-LQ]

Checklist before submitting the issue:

• The issue is strongly related to the MiXCR software
• The issue can be reproduced with the most recent version of MiXCR
• There is no answer to the question in the official documentation and there is no duplicate issue in the bug tracker
• Inspection of raw alignments with exportAlignmentsPretty shows that data has the expected architecture, and sample preparation artefacts are not the reason of the problem (if this is the matter of the issue)

Dear MiXCR team,

I am processing 10X scBCRseq data using the 10x-sc-xcr-vdj preset on multiple samples. The pipeline ran normally for all other samples except this one, where it hit an error on the assembleCells step. I have attached report files for the previous steps for context.

Thank you for your support.
Khang.

Exact MiXCR commands

/fp/projects01/ec195/storage/khangl/software/jdk-22.0.1/bin/java -jar /fp/projects01/ec195/storage/khangl/software/mixcr-4700/mixcr.jar analyze --force-overwrite 10x-sc-xcr-vdj \ --species hs \ --rna \ --drop-non-CDR3-alignments \ --impute-germline-on-export \ /fp/projects01/ec195/storage/khangl/Sequencing_rawdata/250408_Vaxpred_scseq_large/vaxpred_s4_fastq/Reaction03/VDJ/{{a}}_L{{n}}_R1_001.fastq.gz /fp/projects01/ec195/storage/khangl/Sequencing_rawdata/250408_Vaxpred_scseq_large/vaxpred_s4_fastq/Reaction03/VDJ/{{a}}_L{{n}}_R2_001.fastq.gz \ /fp/projects01/ec195/storage/khangl/Sequencing_rawdata/250408_Vaxpred_scseq_large/vaxpred_s4_fastq/Reaction03/VDJ/Reaction03 \ --append-export-clones-field -vGene \ --append-export-clones-field -dGene \ --append-export-clones-field -jGene \ --append-export-clones-field -cGene \ --append-export-clones-field -nFeature VDJRegion \ --append-export-clones-field -aaFeature VDJRegion \ --append-export-clones-field -aaFeatureImputed VDJRegion \ --append-export-clones-field -allNMutations FR1Begin FR4End \ --append-export-clones-field -allNMutationsCount FR1Begin FR4End \ --append-export-clones-field -allAAMutations FR1Begin FR4End \ --append-export-clones-field -allAAMutationsCount FR1Begin FR4End \ --export-productive-clones-only \

MiXCR report files

mixcr assembleCells
Running:
mixcr assembleCells -f --report /fp/projects01/ec195/storage/khangl/Sequencing_rawdata/250408_Vaxpred_scseq_large/vaxpred_s4_fastq/Reaction03/VDJ/Reaction03.assembleCells.report.txt --json-report /fp/projects01/ec195/storage/khangl/Sequencing_rawdata/250408_Vaxpred_scseq_large/vaxpred_s4_fastq/Reaction03/VDJ/Reaction03.assembleCells.report.json /fp/projects01/ec195/storage/khangl/Sequencing_rawdata/250408_Vaxpred_scseq_large/vaxpred_s4_fastq/Reaction03/VDJ/Reaction03.contigs.clns /fp/projects01/ec195/storage/khangl/Sequencing_rawdata/250408_Vaxpred_scseq_large/vaxpred_s4_fastq/Reaction03/VDJ/Reaction03.assembledCells.clns
Initialization: progress unknown
Parsing input: 100%
Grouping clones: 2.9%
Grouping clones: 117.2% ETA: 00:00:00
Overlapping leftover cells barcodes with found groups: 63.6%
Overlapping non functional clones with groups: 23.6%
Overlapping non functional clones with groups: 100% ETA: 00:00:00
Please copy the following information along with the stacktrace:
Version: 4.7.0; built=Wed Aug 07 21:19:48 CEST 2024; rev=976ba14139; lib=repseqio.v5.1
OS: Linux
Java: 22.0.1
Abs path: /fp/projects01/ec195/storage/khangl/Sequencing_rawdata/250408_Vaxpred_scseq_large/vaxpred_s4_fastq/Reaction03/VDJ
Cmd args: assembleCells -f --report /fp/projects01/ec195/storage/khangl/Sequencing_rawdata/250408_Vaxpred_scseq_large/vaxpred_s4_fastq/Reaction03/VDJ/Reaction03.assembleCells.report.txt --json-report /fp/projects01/ec195/storage/khangl/Sequencing_rawdata/250408_Vaxpred_scseq_large/vaxpred_s4_fastq/Reaction03/VDJ/Reaction03.assembleCells.report.json /fp/projects01/ec195/storage/khangl/Sequencing_rawdata/250408_Vaxpred_scseq_large/vaxpred_s4_fastq/Reaction03/VDJ/Reaction03.contigs.clns /fp/projects01/ec195/storage/khangl/Sequencing_rawdata/250408_Vaxpred_scseq_large/vaxpred_s4_fastq/Reaction03/VDJ/Reaction03.assembledCells.clns
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Reaction03.align.report.txt
Reaction03.assemble.report.txt
Reaction03.assembleContigs.report.txt

[mizraelson]

Hi, can you try adding: -MassembleCells.algorithm.thresholdForAssigningLeftoverCells=null
Let em know if it solves the issue

[Khang-LQ]

Hello again,

I tried rerunning the analyze step with -MassembleCells.algorithm.thresholdForAssigningLeftoverCells=null, but the same issue persists, it is giving the same error "picocli.CommandLine$ExecutionException: Error while running command assembleCells java.lang.IllegalStateException: Check failed." in the assembleCells step.

/fp/projects01/ec195/storage/khangl/software/jdk-22.0.1/bin/java -jar /fp/projects01/ec195/storage/khangl/software/mixcr-4700/mixcr.jar analyze --force-overwrite 10x-sc-xcr-vdj -MassembleCells.algorithm.thresholdForAssigningLeftoverCells=null \
--species hs \
--rna \
--drop-non-CDR3-alignments \
--impute-germline-on-export \
/fp/projects01/ec195/storage/khangl/Sequencing_rawdata/250408_Vaxpred_scseq_large/vaxpred_s4_fastq/Reaction03/VDJ/{{a}}_L{{n}}_R1_001.fastq.gz /fp/projects01/ec195/storage/khangl/Sequencing_rawdata/250408_Vaxpred_scseq_large/vaxpred_s4_fastq/Reaction03/VDJ/{{a}}_L{{n}}_R2_001.fastq.gz \
/fp/projects01/ec195/storage/khangl/Sequencing_rawdata/250408_Vaxpred_scseq_large/vaxpred_s4_fastq/Reaction03/VDJ/Reaction03 \
--append-export-clones-field -vGene \
--append-export-clones-field -dGene \
--append-export-clones-field -jGene \
--append-export-clones-field -cGene \
--append-export-clones-field -nFeature VDJRegion \
--append-export-clones-field -aaFeature VDJRegion \
--append-export-clones-field -aaFeatureImputed VDJRegion \
--append-export-clones-field -allNMutations FR1Begin FR4End \
--append-export-clones-field -allNMutationsCount FR1Begin FR4End \
--append-export-clones-field -allAAMutations FR1Begin FR4End \
--append-export-clones-field -allAAMutationsCount FR1Begin FR4End \
--export-productive-clones-only \

I have also tried running assembleCells alone using
/fp/projects01/ec195/storage/khangl/software/jdk-22.0.1/bin/java -jar /fp/projects01/ec195/storage/khangl/software/mixcr-4700/mixcr.jar assembleCells -f -MassembleCells.algorithm.thresholdForAssigningLeftoverCells=null --report /fp/projects01/ec195/storage/khangl/Sequencing_rawdata/250408_Vaxpred_scseq_large/vaxpred_s4_fastq/Reaction03/VDJ/Reaction03.assembleCells.report.txt --json-report /fp/projects01/ec195/storage/khangl/Sequencing_rawdata/250408_Vaxpred_scseq_large/vaxpred_s4_fastq/Reaction03/VDJ/Reaction03.assembleCells.report.json /fp/projects01/ec195/storage/khangl/Sequencing_rawdata/250408_Vaxpred_scseq_large/vaxpred_s4_fastq/Reaction03/VDJ/Reaction03.contigs.clns /fp/projects01/ec195/storage/khangl/Sequencing_rawdata/250408_Vaxpred_scseq_large/vaxpred_s4_fastq/Reaction03/VDJ/Reaction03.assembledCells.clns

but I encoutered this error "Unknown option: '-MassembleCells.algorithm.thresholdForAssigningLeftoverCells=null"

I'm not sure whether the command syntax is wrong or is the option not doing anything to resolve the error?